The invention relates generally to chemical functionalization of surfaces to modify the properties thereof. In particular, the invention relates to improved methods of functionalization of a substrate with silane mixtures to reduce surface energy and constrain droplets of liquid that are applied to the substrate surface. A primary use of the invention is in the field of solid phase synthesis of oligomer arrays.
Functionalized solid surfaces are useful in both chemistry and biotechnology. One important application is in solid phase chemical synthesis, wherein the initial derivatization of a substrate surface allows for synthesis of polymers such as oligonucleotides and peptides on the substrate itself. In addition, support bound oligomer arrays are particularly useful for screening studies and sequence identification in complex nucleic acids. Modification of surfaces for use in chemical synthesis has already been described, for example, in U.S. Pat. No. 5,624,711 to Sundberg et al., in U.S. Pat. No. 5,266,222 to Willis et al., and in U.S. Pat. No. 5,137,765 to Farnsworth.
A number of techniques have been used for modifying siliceous or metal oxide surfaces. One of these techniques includes the derivatization of surfaces with bifunctional silanes, i.e., silanes having a first functional group that enables covalent binding to [a] the surface of interest (often a Si-halogen or Si-alkoxy group, as in xe2x80x94SiCl3 or xe2x80x94Si(OCH3)3, respectively) and a second functional group that can impart the desired chemical and/or physical properties to the surface. However, this type of surface modification often imparts unwanted physical properties to the surface due to the presence of the second functional group. It is currently of interest to produce arrays that may use different oligonucleotides on siliceous surfaces and that have high density of features. The various features can be independently created by the planar separation of individual phosphoramidite coupling reactions as the oligonucleotides are synthesized. This can be most easily accomplished by simply spotting the phosphoramidite solutions onto the surface. Feature density can then be determined by the spread of the solution droplet deposited on the surface, which is determined by the volume of the droplet and the contact angle between the droplet and the surface. However, covalently coupling the first nucleotide phosphoramidite to the substrate surface requires hydroxyl moieties on the surface, which makes the surface wettable by the phosphoramidite solutions and thus creates droplet spread. This limits the quality of the features and the feature density that may be used.
The above mentioned problem can be overcome using a variety of techniques that are well known in the art. For instance, one solution has been to implement the use of permanent wells. Permanent wells can be formed by micro-machining the substrate, with the active surfaces subsequently modified and, therefore, constraining the droplet by the capillary action.
Another method to overcome this problem involves the use of a temporary well that can be formed as a pre-formed stencil or by applying a coating to the substrate and patterning the coating. Alternatively, as described in U.S. Pat. No. 5,474,796 to Brennan, a pattern of two different surface-bound silanes can be formed by physically masking the surface, depositing a first silane, and then removing the mask and depositing a second silane. More recently, techniques have been developed using various derivatizing compositions containing different silanes under anhydrous reaction conditions to provide functionalized surfaces on substrates. As described above, the presence of water, due to addition of aqueous solutions or caused by failing to maintain anhydrous conditions, prevents from obtaining substrates with a functionalized surface and low surface energy. In array fabrication, it is desirable to produce functionalized substrate surfaces without the need to maintain completely anhydrous conditions. It is also desirable to develop techniques that may actually be conducted under aqueous conditions to produce functionalized substrates of low surface energy that are useful for construction of stable array platforms.
The invention provides a process for preparing a substrate having a functionalized surface and a low surface energy. The process comprises contacting a solid substrate having reactive moieties on the surface with a mixture of organic solvent and water, and with a derivatizing composition.
The invention provides a derivatizing composition comprising a first silane, R1xe2x80x94Si(RLRXRY) and a second silane, R2xe2x80x94(L)nxe2x80x94Si(RLRXRY). This derivatizing composition can be used to functionalize a substrate surface that has been contacted with a mixture of organic solvent and water. The RL moieties, which may be identical or different, are leaving groups, RX and RY, which may be identical or different, are either leaving groups, like RL, or are lower alkyl, R1 is a chemically inert moiety that lowers the surface energy of the substrate, n is 0 to 1, L is a linking group, and R2 is CHxe2x95x90CH2. The ratio of the silanes in the derivatizing composition determines the surface energy of the functionalized substrate and the density of molecular moieties that can ultimately be bound to the surface substrate.
In another embodiment, a process is provided for preparing support-bound cleavable ligands on a substrate of low surface energy. The process involves contacting a substrate having reactive moieties on the surface thereof with a derivatizing composition comprising a first silane R1xe2x80x94Si(RLRXRY) and a second silane R2xe2x80x94(L)nxe2x80x94Si(RLRXRY) as discussed above, under reaction conditions effective to couple the silanes to the substrate surface (i.e., the reaction can be carried out by first contacting the substrate surface with a mixture of organic solvent and water and then adding the derivatizing composition or by contacting the substrate surface with the mixture and the derivatizing composition). The functionalization provides xe2x80x94Sixe2x80x94R1 groups and xe2x80x94Sixe2x80x94(L)nxe2x80x94R2 groups on the surface of the substrate. A ligand is then coupled to the surface at R2, through a linking moiety containing a cleavable site. The ligand may be, for example, a small molecule, a first monomer in the solid phase synthesis of an oligomer, an intact oligomer, or the like.
In an additional embodiment, a derivatizing composition is provided for carrying out the aforementioned processes. The derivatizing composition comprises a mixture of silanes, including a first silane R1xe2x80x94Si(RLRXRY) and a second silane R2xe2x80x94(L)nxe2x80x94Si(RLRXRY), wherein R1, R2, RL, RX, RY and n are defined as above.
Finally, the functionalized substrates provided using the presently disclosed and claimed processes and compositions represent a further embodiment of the invention. The substrates have surface-bound xe2x80x94Sixe2x80x94R1 groups and xe2x80x94Sixe2x80x94(L)nxe2x80x94R2 groups, wherein the R1 moieties reduce surface energy and the R2 moieties comprise either functional groups enabling covalent attachment of a molecular moiety of interest or modifiable groups that can be converted to such functional groups.